Fusarium head blight (FHB) is a devastating disease of wheat. Molecular mapping led to the identification of two major FHB resistance QTL, Fhb1 and Qfhs.ifa-5A. The actual function of these resistance genes is still unknown. The resistant line CM82036, the susceptible line Remus and two sister lines from the cross CM82036/Remus were analysed for gene expression. The sister lines show contrasting levels of FHB resistance due to the presence or absence of resistance alleles at Fhb1 and Qfhs.ifa-5A. At anthesis plants were challenged by Fusarium graminearum or water under controlled conditions. At six-time points after inoculation (0-72 h) gene expression of specific wheat floral tissue was analysed by cDNA-AFLPs in two biological replications. Altered expression patterns after F. graminearum inoculation were observed for 164 transcript-derived fragments (TDFs), corresponding to 3.4% of the analysed fragments. Fourteen TDFs, 0.28% of the total analysed fragments, displayed differential expression after fungal attack depending on the genotype; five of these TDFs were differentially expressed between the sister lines and are possibly associated with the possession of Fhb1 and Qfhs-ifa-5A and the FHB resistance level of the genotypes. Sequencing and annotation of these gene tags revealed homologies to a UDP-glucosyltransferase, phenylalanine ammonia-lyase, Dna-J like protein, pathogenesis-related family protein and to one gene with unknown function providing initial clues for guiding further functional studies on the resistance reaction of wheat against FHB. This work is the first report on differential gene expression between related, resistant and susceptible, wheat lines after F. graminearum attack.